Brain Research, 152 (1978) 265 282 (~ Elsevier/North-Holland Biomedical Press

265

I N T R A C E L L U L A R H O R S E R A D I S H PEROXIDASE INJECTION FOR CORR E L A T I O N OF L I G H T A N D E L E C T R O N MICROSCOPIC A N A T O M Y W I T H SYNAPTIC P H Y S I O L O G Y OF C U L T U R E D MOUSE SPINAL CORD NEURONS

ELAINE A. NEALE, ROBERT L. M A C D O N A L D and PHILLIP G. NELSON

Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014 and Department of Neurology, University of Virginia, Charlottesville, Va. 22901 (U.S.A.) (Accepted December 29th, 1977)

SUMMARY

Synaptic interactions between spinal cord neurons grown in dissociated cell culture were studied electrophysiologically, and presynaptic cells were subsequently injected by intracellular iontophoresis with horseraclish peroxidase (HRP). Following histochemical processing, injected cells were filled with dense reaction product which facilitated the light and electron microscopic identification of the individual physiologically typed neurons. This technique applied to neurons in monolayer culture allowed the visualization of complex intercellular relationships in essentially two dimensions. The number and distribution of morphologically defined synaptic contacts was determined for correlation with individual evoked postsynaptic potentials. HRP-filling of inhibitory and excitatory neurons revealed differences with respect to cellular geometry, axonal projection, and the number, location and ultrastructure of synaptic contacts.

INTRODUCTION

Dissociated cell cultures obtained from mouse spinal cord contain neurons of varied morphology 34. Both excitatory and inhibitory synaptic connections have been documented with intracellular recordings from cultured neurons 33 and electron microscopy of these cells has revealed a number of distinct morphologic classes of synapses 27,2s (and M. Henkart, in preparation). In order to determine the structural correlates of a synaptic interaction, both the pre- and postsynaptic cells must be clearly visible and unambiguously identified. Dissociated cell cultures, in which anatomical relationships approach a two-dimensional organization, afford maximum

266 opportunity to view living neurons during physiologic study. However, additional data on neuronal structure are required for certain types of studies. For example, a detailed analysis of the passive membrane properties of an individual neuron requires exact information concerning its geometrical configuration including its distal dendritic processes, while comprehensive study of synaptic transmission between neuronal pairs requires precise knowledge of the spatial distribution of terminals of presynaptic origin on the postsynaptic cell. Furthermore, in order to relate synaptic fine structure with a particular physiologic action, it is necessary to discriminate in the electron microscope those terminals originating from a documented presynaptic cell. To obtain the anatomical information for these studies, we have combined intracellular recording from pairs of cultured neurons with injection of horseradish peroxidase (HRP) into the presynaptic cells. Preliminary reports 2z,24 of successful staining in intact nervous systems of selected individual neurons including their axons and synaptic terminals by intracellular injection of HRP have been followed rapidly by studies from several laboratoriesS,7-9,15,1s-2°,2~',25,z5,36. The use of HRP for labeling cultured neurons further allows one to (1) map patterns of synaptic connectivity among cells, (2) study the gross morphology of neurons without using serial reconstruction techniques, (3) visualize the distribution of labeled synapses on specifically documented postsynaptic cells, and (4) analyze the structure of a given cell in both the light and electron microscopes using routine methods of sample preparation. Ultimately, it is possible to relate this structural information to electrophysiologic observations made on the same cells. In this study, we present the light microscopic anatomy and synaptic fine structure of two HRP-injected spinal cord neurons and electrophysiological recordings from those neurons, one inhibitory and the other excitatory, and from identified cells which were postsynaptic to the injected cells. Since the HRP-labeled presynaptic swellings contacting postsynaptic cells can be enumerated with light microscopy and such swellings studied further with the electron microscope, certain correlations can be made between number of swellings and size of recorded postsynaptic potentials, and between synaptic fine structure and physiologic action. METHODS

Electrophysiology Spinal cords from 12.5-14-day-old fetal mice were mechanically dissociated and plated on 35 mm Falcon dishes as described elsewhere 3~, and cultures were maintained in incubators at 35-36 °C for at least 5 weeks prior to electrophysiological study. Large diameter (20-50 #m) multipolar spinal cord (SC) neurons were penetrated under direct vision on a modified stage of an inverted phase contrast microscope, where the temperature of the culture was maintained at 35 °C. Microelectrodes (50-150 M f~) filled with a 4 ~ solution of HRP (Sigma, Type VI) in 0.05 M Tris.HC1, pH 8.6, containing 0.2 M KC1, were used to impale presynaptic cells; 4 M potassium acetate microelectrodes (20-40 Mr2) were used to record from postsynaotic cells.

267 A conventional bridge circuit allowed simultaneous recording of membrane potential and injection of current through a single microelectrode. For recording, cultures were placed in normal growth medium (90 % Modified Eagle's Medium/lO% horse serum) to which MgC12 and CaClz were added to achieve final divalent cation concentrations of 8-10 mM. The high Mg 2+ concentration decreased spontaneous spike activity, permitting a clear recording of the evoked PSPs which were maintained by the high Ca 2"-. HRP was ejected by 500 msec positive square wave pulses of 5-20 nA at I pulse/ sec for 5-30 min. After physiological study and injection with HRP, the cells were photographed in the living state using phase optics, and the cultures were returned to the incubator for 4-12 h before processing.

Tissue processing Fixation was accomplished by the dropwise addition to the culture medium of an equal volume of 2.5 % glutaraldehyde in 0.15 M sodium cacodylate, pH 7.434; the fixative solution was replaced twice. After an hour at room temperature, the cultures were rinsed several times with buffer and placed in the refrigerator overnight. Demonstration of peroxidase activity was carried out according to Graham and Karnovsky11; the diaminobenzidine (DAB)-hydrogen peroxide reaction mixture was changed 3 times during 15 rain at room temperature. Cultures were rinsed well and postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate, pH 7.4, for an hour in the cold. Following sequential rinses in 0.1 M sodium cacodylate, pH 7.4, and 0.05 M sodium acetate, pH 5.0, the cultures were stained with 1% uranyl acetate in the sodium acetate buffer for an hour in the cold. Dehydration in a graded alcohol series followed, with treatment in absolute alcohol limited to 20 min. Two 10-min changes of a 1:1 mixture of Epon:absolute alcohol and one 10-min exposure to a 3:1 mixture were followed by several changes of complete Epon, before a final layer of Epon, approximately 1 mm thick, was applied over each culture. Such cultures were stored overnight at room temperature in a slightly evacuated desiccator before curing on a level surface at 60 °C.

Light microscopy After curing, but while still warm, the sides of the plastic culture dish were broken away, and the Epon disc containing the cultured cells was gently lifted from the flat surface of the dish. After filing the ragged edges at the circumference of the disc, a coverslip was placed over a drop of water applied to the 'cell' surface, and the preparation handled as a microslide. Using a system of co-ordinates, it was always possible to relocate those cells studied physiologically. Photomicrographs were taken on a Zeiss Photomicroscope II using a blue filter (Zeiss 46 78 50), and various phase, bright-field and dark-field optical systems.

Electron microscopy Fields of interest, 3 0 0 4 0 0 # m on a side, were scored with a needle mounted on a micromanipulator. Areas containing the scores were removed with a jeweler's

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Fig. 1. A: phase contrast photomicrographs of living spinal cord neurons in culture showing cells from which intracellular transmembrane potential recordings were obtained. B: intracellular recordings obtained from synaptically connected cells I and IIa of A. Stimulation of cell I by intracellular current injection produced an action potential in cell ! (upper traces) followed by a hyperpolarizing IPSP in cell IIa (lower traces). Two traces are shown superimposed, one with and one without the stimulus. Calibration pulses represent 10 mV and 2 msec. Resting potential of cell I was --35 mV and of cell IIa was I 6 3 mV. C: IPSPs recorded in cell IIa at different steady membrane potential levels as labeled. Note inversion of the synaptic potential when the steady membrane potential was increased. Calibration pulse in upper trace represents 10 mV and 10 msec. D: an action potential elicited in cell IIa (lower trace) caused a large excitatory postsynaptic potential (EPSP) in cell I. When this EPSP elicited an action potential in cell I (sharp depolarizing event at crest of slower potential waveform), a corresponding IPSP occurred in cell IIa. Two superimposed traces are shown for each cell. Calibration pulses represent 10 mV and 10 msec.

269 saw from the Epon disc, mounted on Epon blanks, and fine-trimmed to the score marks. Serial sections were cut parallel to the growth surface. Short ribbons of sections were collected on formvar coated single-hole (1 × 2 mm) copper grids, stained with uranyl acetate and lead citrate, and examined in a JEOL 100B electron microscope at 80 kV. Low magnification photomontages of postsynaptic cells in serial sections were constructed in order to confirm the spatial correspondence of labeled boutons with swellings seen with light microscopy. RESULTS Phase contrast photomicrographs of cells studied physiologically are presented in Fig. 1A. Stimulation of Neuron I elicited an IPSP (inhibitory postsynaptic potential) in both Neurons IIa (Fig. 1B) and Ilb. The latency fiom the peak of the action potential in Neuron I to the onset of the IPSP in IIa was 1.5--2.0 msec. The IPSPs elicited in IIa and IIb were of a simple monophasic character, and could be readily reversea by polarizing currents passed through the recording electrode in these cells. The reversal potential was about - - 8 0 mV (Fig. 1C) and no excitatory component was revealed by the polarization procedure. It is a finding important for subsequent interpretations that the monosynaptic latency in these cultures may be greater than 1.5 msec as shown in Fig. lB. The H R P injection revealed a massive connection from Neuron I to Neurons IIa and IIb (see below) and clearly the only synaptic response was a simple monophasic inhibitory potential that we interpret as a direct monosynaptic connection. Any alternative interpretation involving an inbibitory interneuron would require that the demonstrated large anatomical connection from I to IIa were non-functional. These latencies of 1.5-2.5 msec indicate that the conduction velocities of the axonal processes involved in synaptic connections between the cells are very low, in the range of 1 meter/ sec or even less. The extremely fine caliber of these unmyelinated fibers (e.g. Fig. 5C) is consistent with this slow conduction velocity. Neurons IIa and IIb were excitatory to Neuron I and the EPSP elicited in Neuron I by an action potential in Neuron IIa was very large, over 40 mV (Fig. l D). When this EPSP produced an action potential in Neuron I, a corresponding IPSP was seen in Neuron IIa. Following the physiologic observations, Neuron I was injected (see Methods) with 5 nA positive current pulses for 35 rain and the culture was fixed 8 h later. The gross morphology of the injected neuron is quite complex (Fig. 2A) relative to the structure visible with phase microscopy. Many processes appear to emerge from the soma; individual processes cannot be easily traced. Within the mat of neurites surrounding the soma, some, possibly dendrites, are considerably more spiny than others. Those processes more distant from the cell body display smooth contours and are presumably axonal branches. Many fine processes emerge from these axon branches, giving rise to terminal swellings. Numerous, but not all, cells in proximity to cell I are heavily encrusted with swellings of these delicate processes. Cell IIa, demonstrated physiologically to be postsynaptic to cell I, is shown in Fig. 2B;

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271 hundreds of labeled swellings are seen to lie along its processes and entwine its soma. Labeled processes, varying in appearance from thick tubes to delicate threads, are seen in this field. The same area sectioned for electron microscopy is shown in Fig. 2C. The surface of cell IIa is laden with synaptic boutons of various morphologies. Those terminals arising from the HRP-injected cell I are easily discriminated (Fig. 3), however, by the increased density of the plasmalemma and synaptic vesicle membranes and by occasional flakes of reaction product throughout the ground substance (Fig. 3B). In this cell, the H R P label is relatively heavy, yet details of presynaptic fine structure remain clearly visible. In the terminals of the injected cell, vesicles are somewhat pleomorphic and are dispersed rather uniformly. Synaptic complexes, limited by arrowheads in Fig. 3B, are distinguished as regions where pre- and postsynaptic membranes parallel each other, the interspace showing evidence of finely filamentous material. Vesicles and occasional tufts of dense material adhere to the presynaptic membrane at these sites, although postsynaptic density is generally not striking. Several synaptic complexes are commonly seen in one bouton. The axon of Neuron IIa was distinguishable in the electron microscope for approximately 125 ffm after it emerged from the base of a dendrite; there were no HRP-labeled boutons contacting it over this length. No ultrastructural differences were evident among the labeled terminals impinging on the dendrites or somas of 3 different postsynaptic cells examined. Excitatory synaptic transmission was observed quite frequently in these experiments, with the presynaptic cell usually contacting several neurons. The neuron labeled I (Fig. 4A) was presynaptic and excitatory to those neurons labeled IIa and IIb. The initial resting membrane potential of cell IIa was somewhat unstable and low, - - 3 5 to - - 4 0 mV, and the EPSP was only about 2 mV. When the membrane potential was increased to a b o u t - - 1 4 0 mV by steady hyperpolarizing current, the EPSP increased to 4 mV (Fig. 4B) but, due to the unsatisfactory recording, a reliable extrapolated reversal potential could not be obtained. The recording from cell lib was technically more satisfactory with a stable membrane potential o f - - 6 0 mV and 3.5 mV EPSP (Fig. 4C). The extrapolated reversal potential for the EPSP recorded in IIb was - - 2 0 mV, somewhat more negative than in earlier studies of the SC-SC EPSP 3~. In addition to the early monosynaptic EPSP, a late polysynaptic excitatory wave was seen in both cells IIa and b when multiple action potentials were elicited

Fig. 2. Bright-field photomicrographs of the same cells shown in Fig. 1, after intracellular iontophoresis of HRP into Neuron I, fixation, enzyme incubation and processing for electron microscopy. A: low power photomontage of the HRP-filled inhibitory cell and its processes. The dome-shaped cell body is surrounded by a tangle of neurites, while numerous smooth processes, presumably axonal branches, radiate greater distances. Thick processes give rise to progressivelymore delicate branches which, in turn, form terminal swellings. Many cells in the vicinity of the stained neuron, including Neurons IIa and lib, appear peppered with these swellings, x 120. Bar -- 200 ffm. B: high magnification view of Neuron IIa, whose polygonal cell body and dendritic trunks are studded with labeled swellings. Numerous HRP-filled processes traverse the field. Arrows indicate those swellings shown in electron micrographs in Fig. 3. x 790. Bar -- 50 fire. C: an ultrathin section of the area selected for electron microscopic examination, x 100.

272

273 in N e u r o n I. The monophasic EPSPs elicited in IIa and b were presumably m o n o ;ynaptic, since the latency was about 2 msec, too short to allow an intercalated interneuron to be involved (see above). Following physiological study, cell I was injected with H R P for 30 min using 12 n A positive pulses of 500 msec duration at a rate of 1/sec, and the culture was fixed 9 h later. The same field, after histologic processing and embeclding, is shown in Fig. 5A. The soma of the presynaptlc injected cell is clearly visible. Processes, presumed to be dendrites, emerge from the cell b o d y at several locations, show tapering diameters and minimal branching, and extend for up to 350 # m from the cell body. One process, t h o u g h t to be the axon (ax), shows a rather constant diameter, and can be followed for approximately 2.5 m m where it becomes only faintly visible. In contrast to the dendrites, the contours of the axon are s m o o t h and regular. Several fine collaterals < 1 # m in diameter leave the axon, and the point of exit of one of particular interest is shown in the inset. This slender process (small arrows) courses for 350 # m before it contacts cell I I a and then cell IIb; it travels back a m o n g the injected dendrites and finally terminates on a cell (not shown) above tbe injected neuron. The collateral is visible for a total o f 1700 #m. Selected interactions of this collateral with cells I I a and l i b are shown in Fig. 5B and C. The collateral itself exhibits 'en passant' varicosities (asterisks). In addition, an even finer process emerges from the collateral (at arrow, Fig. 5C) and gives rise to numerous 'en passant' swellings lying along the processes o f cell lib. Continuity between the labeled swellings and with the collateral in Fig. 5B presumably exists, but could not be resolved. Between 25 and 30 collateral swellings on the surface of cell IIa were detected by light microscopy; a similar n u m b e r was found on cell lib. That these swellings correspond to synapses was documented with the electron microscope. The area scored for examination included cells IIa and l i b ; a representative thin section is shown in Fig. 6A. A l t h o u g h the peroxidase labeling of such fine processes at some distance from the cell b o d y is very light, labeled terminals can be positively identified. Reaction p r o d u c t tends to accentuate membranes, as illustrated by Fig. 6B, in which a labeled and non-labeled b o u t o n are seen side by side. Due to the light HRP-labeling of these terminals, care was taken to m a p each terminal t h r o u g h serial sections and to confirm its spatial correspondence to a collateral swelling seen with the light microscope. The labeled collateral itself is clearly discriminated, tor the reaction p r o d u c t also adheres to neurotubules and neurofilaments (Fig. 6C and D). Frequently it was possible to trace a labeled process t h r o u g h serial sections as a means of locating more subtly labeled terminals. The 'en passant' b o u t o n denoted

Fig. 3. Electron micrographs of HRP-labeled synaptic terminals on the surface of Neuron IIa, shown to receive inhibitory input from the HRP-injected neuron. A: labeled boutons (arrows) are easily discerned from among the many swellings that cover essentially the entire surface of Neuron IIa. The HRP-reaction product accentuates membranes and increases the electron density of the synaptic cytoplasm. Unlabeled boutons of differing morphologies are seen adjacent to the two that are labeled. × 26,000. Bar -- 1 #m. B: part of a labeled terminal which contains rather pleomorphic vesicles and a large number of mitochondria. Synaptic complexes are limited by arrowheads. Tufts of presynaptic dense material are frequent; postsynaptic density is minimal. × 65,350. Bar -- 0.5/~m.

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275 by the asterisk in Fig. 6C corresponds to the asterisk in Fig. 5B; the double asterisks likewise correspond. The terminal shown in Fig. 6D is that noted with the asterisk in Fig. 5C. Of the 30 swellings along the soma and dendrites of cell IIb, 19 were included within tbe sections studied. It was possible to relocate 15 of those swellings, each of which corresponds to a labeled bouton containing numerous vesicles. The plane of section allowed visualization of synaptic complexes in 11 of the terminals. In the terminals arising from the collateral, primarily round vesicles tend to be clustered near synaptic membranes. Tufts of presynaptic dense material are a common feature of the synaptic complex, while the presence of subjacent postsynaptic density is variable. Four boutons were examined in 5-8 serial sections. The linear extent of the apposition between pre- and postsynaptic elements was measured, as was the length of each identifiable synaptic complex. It was found that individual complexes were not necessarily continuous throughout the sections studied, and that the complexes in one terminal, taken together, comprised approximately 25 ~ of the length of apposition. There were from 2 to 5 discrete synaptic complexes/bouton in the terminals examined. DISCUSSION Although various methods for the selective labeling of neurons are available6,10, 17,37, the technique of intracellular injection of H R P 25 offers the strong advantages of ease and applicability for both light and electron microscopy with good preservation of cellular ultrastructure. The use of this technique in a tissue culture system further allows a morphologic study in which the presynaptic and postsynaptic cells in a synaptically connected network can be definitively identified and visualized in two dimensions. Viewed with bright-field microscopy, successfully injected cells appear in striking contrast to a background of cells lightly stained as a result of endogenous oxidase and osmium treatment. The processes of a given cell can be tentatively identified as axonal or dendritic. It has been possible to trace axonal processes for 15 mm 36 (3.5 mm in this study), and to detect stained processes of only 0.25 # m in diameter. In addition, the synaptic terminals of the injected cell can be identified in the light and electron microscopes, allowing a full characterization of the terminal distribution on postsynaptic cells. The precise parameters for a successful injection in this system are still under study. We have attempted to inject a total of 80 cells using currents ranging from

Fig. 4. A: phase contrast photomicrographs of living synaptically connected neurons from which intracellular transmembrane potential recordings were obtained. B: action potential produced in cell I of A (trace B1) by a current pulse of 0.2 nA (B2) which evoked the EPSP in cell IX a shown in trace B3. The calibration pulses represent 10 mV and 10 msec. C: EPSPs in cell IIb evoked by stimulation of cell I are shown at 3 different steady values of membrane potential produced by long (approx. 60 msec) pulses of current passed across the cell membrane. The resting potential of cell I was --35 mV and of cell IIb was --60 mV. Note the increase in EPSP amplitude with membrane hyperpolarization and decrease with depolarization.

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277 2 to 30 nA and times ranging from 5 to 30 min giving a time-current range of 10 nA/ rain to 900 nA/min. Although most of the injected cells showed some evidence of staining, slightly fewer than half (37 of 80) were well stained and apparently undamaged by the injection procedure. We feel, from recent experiments, that positive 500 msec current pulses (5-10 nA) at l/sec for 5-10 rain (20-50 nA/min)are least injurious to the cells and provide for passage of the enzyme in the presence of 0.2 M KC1. The time course of movement of the injected enzyme within the neuron has not been definitively studied. The possibility exists that diffusion or an active transport could account for the distances labeled. However, Bennett et a l ) have reported that movement of H R P through axoplasm can also occur during fixation. In the present study, cells injected with H R P for 7 rain and fixed 15 min later showed evidence of only light staining ~ 0.5 mm from the cell body while intense staining, 3.5 mm from the cell body, was seen in injected cells allowed to survive 4 h before fixation. Thus, a survival interval appears necessary for more complete enzyme filling of injected cells. The intensity of staining at the light microscopic level varies somewhat from cell to cell and within a given cell. This is reflected by the amount of HRP-reaction product seen with the electron microscope. The reaction product in the cell body and proximal processes is more dense than that seen in more distal processes and terminals. The subcellular distribution of label seen in this study is compatible with that previously reported 7,15,2a, although it appears in some cases (see Fig. 3B) that the reaction product has penetrated and is localized within membranes, and is not simply adherent to the exposed surface. The deposition of reaction product is such, however, that even those boutons barely visible with light microscopy were adequately tagged (Fig. 6) and could be identified as originating from the injected cell, while, in those boutons more heavily labeled, details of synaptic fine structure were not obscured (Fig. 3). The general configuration of these excitatory and inhibitory cells is markedly different. The excitatory axon branches at some distance from the cell body; in contrast, the inhibitory axon ramifies proximally, its branches richly entwined "in the vicinity of the soma. Furthermore, sparse, mainly dendritic, contacts of the excitatory collateral are unlike the very heavy somatic and proximal dendritic inhibitory synaptic investment. Excitatory connections more dense and direct than those shown here would be expected to occur on the basis of such large EPSPs as shown in Fig. 1D,

Fig. 5. Bright-field photomicrographs of the cells in Fig. 4 after HRP injection into Neuron I and subsequent processing. A: short minimally branched dendrites emerge from a polygonal cell soma, and can be distinguished from the long, smooth axon (ax), which courses unbranched for several mm. Small arrows mark a collateral of interest; the inset corresponds to the small box and shows the exit of this collateral from the axon. The boxed areas of Neurons lla and lib, which receive excitatory input from Neuron I, are shown below. × 120. Bar -- 200 /~m. Inset, x 800. B: the HRP-filled collateral passes along Neuron lla; * and ** denote swellings shown in Fig. 6C. C: the collateral gives rise to an even finer branch (at arrow) which forms numerous en passant swellings along the processes of Neuron lib. The asterisk indicates an en passant swelling of the collateral itself and is shown in Fig. 6D. B and C, × 800. Bar = 50/~m.

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279 and indeed we have seen examples of heavy excitatory synaptic investment by several injected excitatory cells. Our experience with inhibitory neurons is not yet extensive. As had been the case in an earlier study 2s, evoked inhibitory synaptic activity, such as that shown in Fig. 1, was observed less frequently than excitatory activity. It m a y be that this relative infrequency of recorded inhibitory synaptic interactions was a result of sampling bias; large cells with prominent processes were selected for physiologic study, and it is possible that many inhibitory cells were thus excluded. It seems likely, on the basis of 4 injected inhibitory cells, that one configuration for inhibitory interneurons is that of a local and extensive ramification of presynaptic processes with a relatively confined field of synaptic activity. Inhibitory neurons with more farreaching processes might also be expected, since interneurons with extended axons, such as those mediating the direct inhibitory interaction between muscle antagonists, have been described 16 in the spinal cord. Anatomical categorization of the different functional synaptic interactions observed in the culture system is in progress. Differences in synaptic morphology were seen in the inhibitory and excitatory terminals. Although the association of elongated vesicles with inhibitory synapses in the spinal cord has been suggested repeatedly4,12, 38, the vesicles in the inhibitory terminals examined were not strikingly pleomorphic. Preliminary measurements, using the parameters defined by Nakajima 26, of the inner diameters of vesicles in the inhibitory (n -- 132) and excitatory (n ~ 110) boutons shown in Figs. 3B and 6D indicate that the 'inhibitory' vesicles are somewhat smaller (mean inner diameter 4- S.D. of 34 nm 4- 80) and slightly more elongated (mean index of elongation 4- S.D. of 1.22 4- 0.2) than those in the excitatory terminal (37 nm 4- 60; 1.10 40.04). When compared using a t-test, the two vesicle populations differ significantly in both parameters with probability values of 0.005 and 0.001 respectively. Although the HRP-labeled inhibitory vesicles studied appear only slightly flattened, unlabeled boutons in the same cultures contain vesicles showing a greater degree of pleomorphism (see Fig. 3A), with elongation indexes of 1.7-1.8. It may be that the very flat vesicles represent an entirely different type of synapse, but it is also possible that the peroxidase or its reaction product has an effect on vesicle shape, perhaps preventing flattening that might occur during fixation ag. However, HRP-injected cells in cultures prepared from cerebellum do contain labeled vesicles that are considerably more pleomorphic than those seen as yet in the studied inhibitory cells in cultures prepared from spinal cord (unpublished observations). More than one type of inhibitory system may de-

Fig. 6. Electron micrographs of HRP-labeled terminals originating from the excitatory Neuron I. A: representative ultrathin section showing area studied. Cells IIa and IIb are discernible; the other dark areas correspond to the cytoplasm of flat cells which cover the surface of the plastic culture dish. × 100, B : adjacent boutons, one of which shows increased electron density of plasmalemma and synaptic vesicle membranes as evidence of HRP-labeling. × 36,050. C: dendrite (den) of neuron IIa receives many synaptic terminals, although those labeled with HRP (* and **, see Fig. 5B) can be discriminated. × 18,600. Bar = 1 /zm. D: en passant swelling of the collateral (* in Fig. 5C). The vesicles within this excitatory bouton are primarily round. A synaptic complex is indicated with arrow heads; pre- and postsynaptic dense material is usually seen. nf, neurofilament; nt, neurotubule. × 65,350. Bar = 0.5/~m.

280 velop in these spinal cord cultures, and further characterization of the pharmacologic properties of the synaptic inhibition, in conjunction with fine structural studies, may be useful in distinguishing between such systems. Regardless of vesicle shape, the two HRP-labeled terminal types shown here can be discriminated from one another (and from a third type originating from an HRP-injected dorsal root ganglion cell; unpublished observations) by additional ultrastructural features; e.g. postsynaptic density, length of junction and vesicle distribution. This study allows some interesting calculations as to the relationship between morphology and function in a central synapse. The probable number of boutons comprising the excitatory connection between cell I and cell IIB (Figs. 4 and 5) is 30, and the total number of presumed release sites is 90-120. Previous calculations 3a (based on analysis of variance of PSPs) had indicated a quantal size for SC-SC excitatory connections of about 200/~V (100-400 #V in various cells). An upper limit of 300-400/*V for quantal size is suggested by the tact that in tetrodotoxin-poisoned preparations, no spontaneous synaptic potentials are seen above the system noise level of 300-500 #V. Thus, either there is no 'spontaneous' unitary synaptic activity, or the quantal size is less than approximately 400/~V. The amplitude of the EPSP evoked in cell IIb by an action potential in cell I was about 3.5 mV. If a quantal size of 200 #V were assumed, the quantal content of this EPSP would be about 18. Since the total bouton count in this connection is 30, the quanta released per bouton would be 0.6, and the quanta released per anatomically defined release site around 0.2. If one quantum corresponds to one vesicle, this means that one stimulus causes the release of one vesicle per 5 release sites. The importance of somatic or dendritic location of synapses as a determinant of the physiologic behavior of central synapses has been extensively discussed1,2, 21, 28,30. Similarly, the electrical properties of neurons have been related to their morphologic characteristicsla,14,31, a2. The available quantitative electrophysiologic techniques can be applied to this neuronal culture system and further correlation can be made with the detailed morphologic description of cellular and synaptic structure provided by H R P injection. Such an analysis is in progress. Studies of neuronal ultrastructure utilizing intracellular iontophoretic injection o f a neuronal marker require cautious interpretation in that the injection procedure involves the passage of large, prolonged depolarizing currents across the cell membrane, so that the ultrastructure of the terminals at the time of fixation may reflect a recovery state. Nevertheless, it was frequently the case that action potentials could be elicited from the presynaptic cells following injection and that these action potentials evoked synaptic potentials in the postsynaptic cells. Since survival intervals were minimally 4 h, the morphology should not reflect an acute poststimulus condition. It will be of interest, however, to compare details of fine structure after iontophoretic injection with those after pressure injection of HRP. Given these reservations, intracellular administration of H R P provides an effective method for structure-function studies in the nervous system. The technique imparts, to selected neurons, adequate contrast over long distances while maintaining cellular integrity so that details of structure may be analyzed at several levels of reso-

281 lution. The increased visibility of individual n e u r o n s afforded by the m o n o l a y e r culture system further increases the potential o f this technique for a n a t o m i c a l studies o f physiologically characterized neurons. ACKNOWLEDGEMENTS We t h a n k S a n d r a C. Fitzgerald, R a y m o n d T. R u s t e n a n d L i n d a M. Bowers for skillful technical assistance.

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Note added in proof. A recent report (Cullheim, S., Kellerth, J. O. and Conradi, S., Evidence for direct synaptic interconnections between cat spinal a-motoneurons via the recurrent axon collaterals: a morphological study using intracellular injection of horseradish peroxidase, Brain Researeh, 132 (1977) 1-10) describes collateral terminations in intact spinal cord similar to the excitatory contacts presented here.